Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Exosomes are 30-100 nm vesicles secreted by a wide range of mammalian cell types (Keller et al. (2006). Immunol. Lett. 107(2): 102-8). An exosome is created intracellularly when a segment of the cell membrane invaginates and is endocytosed (Keller et al. 2006 supra). The internalized segment is broken into smaller vesicles that are subsequently released or shed from the cell. The latter stage occurs when the late endosome, containing many small vesicles, fuses with the cell membrane, triggering the release of the vesicles from the cell. The vesicles (once released are called exosomes) consist of a lipid raft embedded with ligands common to the original cell membrane. Exosomes secreted by cells under normal and pathological conditions contain proteins and RNA molecules including mRNA and miRNA, which can be shuttled from one cell to another, affecting the recipient cell's protein production. However, of importance in the context of their therapeutic and diagnostic utility is the fact that they carry RNA and protein from the cell they were released from. Exosomes appear to lack DNA and possibly ribosomal RNA and contain mostly mRNA and miRNA.
In the late 1970's, tumour-derived exosomes were identified in the peripheral circulation of women with ovarian cancer. Since then, a range of cell types have been demonstrated to be capable of releasing exosomes, including reticulocytes, B cells, T cells, mast cells, epithelial cells and embryonic cells. Exosomes are reported to be secreted in increased amounts in a variety of biological fluids and can be enriched with certain RNA transcripts several hundred-fold compared to normal cells (Hong et al. 2009, BMC Genomics 10:556-569). Since plasma derived exosomes lack ribosomal RNA, RNA extracted from exosomes exhibits a higher proportion of transcripts specific to tumour cells, for example, that may even otherwise be below the detection limit in the tumour cells themselves.
Accordingly, since their discovery, a growing number of therapeutic and diagnostic applications have been developed, in particular in the context of neoplastic conditions. The use of exosomes has garnered considerable interest as vaccine candidates for tumor immunotherapy (Delcayre and Le Pecq, 2006, Curr Opin Mol Ther 8:31-38). Much of this interest stems from the difficulty associated with dendritic cell-based immunotherapy and how an exosome-based approach can overcome some of these difficulties. Tumor cell-derived exosomes containing tumor antigens plus MHC class 1 molecules can transfer tumor antigens to dendritic cells to induce a CD8+ T-cell dependent anti-tumor immune response (Hao et al. Cell Mol Immunol 2006; 3:205-211). Exosomes released from dendritic cells pulsed with tumor antigens were also shown to elicit strong anti-tumor responses. Data obtained in mice have shown that exosomes obtained from dendritic cells pulsed with tumor peptides could prime specific cytotoxic T lymphocytes (CTLs) in vivo and limit or suppress growth of established murine tumors in a T-cell-dependent manner (Zitvogel et al. Nat Med 1998; 4:594-600; Hao et al. Immunology 2007; 120:90-102).
Interestingly, tumor-derived exosomes may have broader activity than previously believed as one study showed that exosomes isolated from different tumors inhibited not only syngeneic but also allogenic tumor growth, indicating that tumor-derived exosomes may harbor some common tumor antigens (Wolfers et al. Nat Med 2001; 7:297-303). Together, these studies indicate that exosomes can be recovered from tumor cells or from dendritic cells pulsed with tumor antigens to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free vaccine.
The successful use of exosomes in cancer immunotherapy has also lead to the hypothesis that they could function as vaccine candidates in the context of infectious diseases. Aline et al. demonstrated that exosomes derived from dendritic cells pulsed with T. gondii tachyzoite sonicates could induce a protective immune response against T. gondii infection. These exosomes primed an antigen-specific cellular and humoral immune response, which provided a good protection against both acute and chronic toxoplasmosis (Aline et al. 2004, Infect Immun. 72:4127-4137). Moreover, CBA/J mice vaccinated with exosomes isolated from T. gondii antigen-pulsed dendritic cells exhibited significantly fewer brain cyst (Beauvillain et al. 2007, Microbes Infect 9:1614-1622).
Another application of exosomes in immunotherapy has been implicated in the treatment of pneumococcal infection in mice (Colino et al. 2007, Infect Immun 75:220-230). Colino and Snapper showed that murine bone marrow-derived dendritic cells (BMDCs) pulsed in vitro with intact diphtheria toxin (DT)-released exosomes, which upon injection into mice induce immunoglobulin G (IgG)2b and IgG2a responses specific for DT (Colino et al 2007, supra). Exosomes have also been evaluated in the context of Streptococcus infections. Invasive strains of Streptococcus pneumoniae are leading causes of meningitis and major causes of otitis media and bacteremia in children and pneumonia in the elderly (Lopez, 2006, Int Microbiol 9:179-190). Vaccine-mediated protection against S. pneumoniae infection is based on humoral immunity specific for S. pneumoniae capsular polysaccharides (Cps) (Makwana et al. CNS Drugs 2007, 21:355-366). Similar to the DT exosomes, BMDCs treated with Cps14 released exosomes enriched in Cps14. These purified exosomes could induce a S. pneumoniae-protective Cps14-specific immunoglobulin M and IgG3 response in naive recipients (Colino et al. 2007, supra).
Exosomes as a vaccine has also been explored for atypical severe acute respiratory syndrome (SARS) caused by the positive-stranded RNA virus, SARS-associated cornavirus (SARS-CoV). Studies by Kuate et al. showed that exosomes containing spike S protein of SARS-CoV induced neutralizing antibody titres (Kuate et al. 2007, Virology 362:26-37). This immune response was further accentuated by priming with the SARS-S exosomal vaccine and then boosting with the currently used adenoviral vector vaccine (Kuate et al. 2007, supra).
In addition to the potential use of exosomes as vaccines against infectious diseases, exosomes have also proved useful in treatment of autoimmune diseases in animal models. This is illustrated in studies by Kim et al. who showed that administration of exosomes derived from dendritic cells-expressing recombinant IL-4 was able to modulate the activity of APC and T cells in vivo, partly through a FasL/Fas-dependent mechanism, resulting in effective treatment against collagen-induced arthritis through suppression of the delayed-type hypersensitivity inflammatory response (Kim et al. 2007, J Immunol. 179:2235-2241).
Exosome display technology is a novel technique of manipulating the molecular composition of the exosomes and tailoring exosomes with new desirable properties. Recently, exosome display was applied for the induction of epitope-specific antibody response against tumor biomarkers (Delcayre et al. Blood Cells Mol Dis 2005, 35:158-168). This technology opens up new possibilities in designing novel therapies and generating new diagnostic tools. Exosome display has been used to prepare recombinant vesicles carrying cytokines as well as tumor antigens that may or may not have been previously found on exosomes (Delcayre et al. 2006 supra). The targeted co-delivery of antigens with the activators of dendritic cells, B-, T- or natural killer cells may also improve the efficacy of exosome-based vaccines.
In terms of diagnostics, the proteins associated with renal diseases could be detected on exosomes isolated from urine, indicating a possible use for urine exosomes as biomarkers (Pisitkun et al. 2006, Mol Cell Proteomics 5:1760-1771). For instance, Pisitkun et al. demonstrated the excretion of exosomes containing aquaporin-2 protein in autosomal dominant and autosomal recessive nephrogenic diabetes insipidus patients (Pisitkun et al. Proc Natl Acad Sci USA 2004, 101:13368-13373). Similar proteomic studies performed on urinary exosomes generated a long list of molecular signatures, illustrating valuable potential for diagnostic, prognostic and pathophysiological discovery (Hoorn et al, 2005, Nephrology (Carlton); 10:283-290).
Similarly to renal pathologies, exosomes are also an attractive source of biomarker candidates for cancer diagnosis including, for example bladder cancer. The differentially expressed proteins found in exosomes include psoriasin, keratin-14, galectin-7, epidermal fatty acid binding protein (E-FABP), migration inhibitor factor-related protein (MRP8) and 14 and stratifin, which may be useful markers for the diagnosis of bladder cancer (Pisitkun et al. 2006, Mol Cell Proteomics 5:1760-1771).
Exosomes may also be valuable as biomarkers or as a source of biomarkers for infectious diseases, for example in the context of defining treatment success. Exosomes may be particularly useful in the context of tuberculosis (TB) as the time required to test a new TB drug treatment protocol is extensive, leading to high drug development cost as well as delays in the introduction of new medication. A major limitation in developing an efficient drug treatment for TB is the lack of available methodology to identify an early infection as well as to determine drug treatment efficacy. Currently, a major goal of TB research is to identify disease biomarkers in biological fluids that can be measured relatively inexpensively for early diagnosis of disease and treatment monitoring.
It has therefore become increasingly clear, as new exosome studies are published, that these small bioactive membranous microvesicles are important in a wide range of biological functions. From their original discovery in the removal of unwanted proteins from maturing reticulocytes to their role in immune surveillance, the inventory of functions continues to grow. As cancer phase I clinical trials have shown, the knowledge of exosomes can be applied therapeutically and the use of exosomes in diagnostics is also likely to grow. However, the absence of standardised and specific methods of exosome recovery as well as exosome-specific quality control methods to maximise the purity of recovered exosome populations has been a significant limitation to furthering the use of exosomes both therapeutically and diagnostically. Current approaches are centred on capturing or enriching for exosomes from plasma using antibody-based capture (targeting exosome specific surface antigens), ultracentrifugation (i.e. centrifugation speed>60,000 g) or filtration. Both immunocapture and ultracentrifugation based isolation of exosomes require advanced equipment instruments and highly skilled staff. Hence these technologies are not readily implemented at clinical sites. Other methods for purifying exosomes are based on two sequential spins of a biological fluid, such as plasma, at 1,600 g each. However, detection and measurement of nucleic acids derived from circulating exosomes requires complete elimination of haematopoietic cells as their cellular. RNA content otherwise may affect the specificity of PCR-based detection of biomarkers of interest. To date, the methods which have been utilised have not achieved particularly good enrichment or purification of exosomes, particularly to the extent that these methods aim to recover and analyse exosome-derived RNA, the result of which is of limited value if contaminating cellular RNA is not removed. Accordingly, there is an ongoing need to develop better methods for isolating exosomes, in particular from plasma.
In work leading up to the present invention it has been determined that a significantly improved level of membranous microvesicle purity in a biological sample, relative to a contaminating cellular population, can be obtained if the biological sample is subjected to mechanical cellular rupture. This is achieved by virtue of the fact that the physical structure and characteristics, such as mass, of a membranous microvesicle have been determined to be sufficiently different from those of a cell that selective disruption of the membrane of a cell, but not a membranous microvesicle, can be achieved. The nucleic acid or proteinaceous material released from the cells is thereafter degraded by the actions of enzymes either naturally present or introduced to the biological sample. This effectively and simply clears the biological sample of non-microvesicle nucleic acid material, thereby achieving enrichment of the membranous microvesicles. Still further, in terms of conducting analysis of the membranous microvesicle-derived nucleic acid or protein material, there is no need to necessarily conduct any further purification or separation steps on the biological sample since the nucleic acid and protein component of the biological material is essentially only the nucleic acid or protein derived from the non-disrupted component of the specimen, that is, the membranous microvesicles. The technique described herein is extremely simple and effective, thereby enabling the enrichment of membranous microvesicles, such as exosomes, relative to a cellular population, from any biological sample, such as blood, plasma, lymph, saliva and urine, where the enriched membranous microvesicles which are obtained are intact and therefore suitable for application in any therapeutic, diagnostic, prognostic or other application.
To the extent that the enrichment of an exosome population is sought for the purpose of extracting and analysing the exosome mRNA, it has been further determined that exosome-derived mRNA does, in fact, comprise a poly(A) tail, thereby enabling very simple isolation of exosome mRNA via the specificity provided by the poly(A) tail. This enables isolation of the mRNA with minimal signal loss. mRNA isolation methods which have been used to date have generally been more complex in that they have been based on less specific means of isolating free nucleic acid molecules, such as silica columns.
Still further, methods of efficiently achieving the recovery and amplification of exosome RNA have been developed. The development of these methods now facilitates the routine recovery of more highly enriched populations of exosomes from a biological sample and further, the RNA contained therein. This has not previously been achievable at the level of simplicity and specificity now provided. The method of the present invention is useful in a range of diagnostic, therapeutic, research and development applications.